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Introduction Salmonella enterica serovar Typhi (S. Typhi) causes enteric fever with >22 million cases and >200,000 deaths worldwide annually 1 . Blood culture is the most widely used diagnostic modality but has limited sensitivity 2 and requires significant resources, limiting its use in resource poor countries where enteric fever is endemic. The lack of an effective diagnostic hampers clinical care, limits disease surveillance efforts and may drive inappropriate antibiotic prescriptions. Detection of the S. Typhi capsular polysaccharide (Vi antigen) in urine could potentially be a useful point-of-care diagnostic. Similar assays are routinely used to detect Legionella and pneumococcal antigens in urine. Attempts to create an Enzyme-Linked Immunosorbent Assay (ELISA) has been previously attempted twice with polyclonal and monoclonal antibodies with mixed results 3-4 . Since 2010, the Oxford Vaccine Group has developed a controlled human infection model of typhoid infection5. From these trials, urine sample were collected longitudinally after S. Typhi challenge. Using these samples, the authors aimed to construct an new ELISA configuration to detect Vi antigen in urine. Methods A sandwich ELISA design was developed using the reagents as demonstrated in figure 1. Using this design, a series of optimization experiments were performed using phosphate buffered solution (PBS) spiked with purified Vi antigen (purified from Citrobacter freundii, National Institute for Biological Standards and Contol (NIBSC), UK). From these, the optimised method for the ELISA was determined as follows: 1. Coat 96 well ELISA plate with 50μl/well of monoclonal IgM (3μg/ml) and incubate at 5 0 C overnight. Wash plate (x5) with PBS/0.05% Tween (PBST) 2. Block each well with 100μl PBS/1% Bovine Serum Album (BSA), incubate at 37 0 C for 2 hours and then wash plate with PBST. 3. Add 50μl sample analyte per well, incubate at room temperature for 2 hours and then wash plate with PBST. 4. Add 50μl polyclonal anti-Vi IgG (1:4000 dilution) per well, incubate at room temperature for 1 hour and then wash plate with PBST 5. Add 50μl conjugated Anti-rabbit IgG (1:12500 dilution) per well, incubate at room temperature for 1 hour and then wash plate with PBST 6. Add 50μl of TMB to each well. Stop reaction with 4M H 2 SO 4 when top of standard curve reads at OD of 0.8-1 at 650nm with the ELISA plate reader After optimisation of the method, optimal standard curves and controls were determined. The ELISA was validated using urine from healthy volunteers spiked with known quantities of Vi-antigen. Stored urine samples from a previous human typhoid challenge model were tested using the ELISA. In the challenge model, participants ingested a 10 4 CFU S. Typhi on day 0 and followed up daily for 14 days. Participants were treated with ciprofloxacin 500mg BD at day 14 or when diagnosed with typhoid infection (defined as blood culture positive and/or temperature ≥38 O C for >12 hours). Urine samples from diagnosed participants at two time points (baseline and day of diagnosis) were tested using this assay. Results The optimisation and validation process produced a ELISA that can detect Vi antigen to a lower detection limit of 0.24 ng/ml. The optimal standard curve by this ELISA is shown in Figure 2. The results of validation of the ELISA using urine spiked with known concentrations of Vi antigen are shown in Figure 3. Urine from 21 participants diagnosed with typhoid from previous human challenge studies were tested using this ELISA (Table 1) Christopher A Darlow 1 , Christina Dold 1 , Malick M Gibani 1 & Andrew J Pollard 1 1 - Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford, UK Discussion This series of experiments has demonstrated a novel ELISA setup that can detect Vi antigen in urine samples spiked with Vi-antigen, with a lower limit of detection at 0.24 ng/ml. However, this assay was unable to detect Vi-antigen in urine samples obtained from human challenge participants with typhoid fever. There are several potential reasons for this. First, the samples from human challenge studies had been centrifuged, filtered and subsequently frozen at -80 0 C. Any of these processing methods could have removed or altered the Vi antigen from samples. Secondly, the assay itself may have an insufficiently low detection limit for Vi antigen, which may require adjustment to the ELISA design. Finally, the human challenge model may not be adequately representative of typhoid patients in the field, as participants are treated very early in the course of infection, when the bacterial load may be too low to produce adequate Vi antigen in the urine. To delineate this further, the authors intend to use the ELISA prospectively on unprocessed urine samples from upcoming human challenge studies. Additional future directions are to test on field samples from typhoid patients or to increase the sensitivity of the ELISA by modifying the assay design, such as using a multimerised HRP-conjugated detection antibody. A New Enzyme-Linked Immunosorbent Assay (ELISA) for Vi antigen as a potential diagnostic for infections by Salmonella Typhi Fig 1 – A schematic depiction of the ELISA setup, with key Fig 2 – Example standard curve produced by the ELISA, with high, medium and low controls using PBS/BSA spiked with Vi antigen Fig 3 – Demonstration of the readings from known concentrations of Vi in Urine (red) against the standard curve (from PBS/BSA spiked with Vi antigen) of the ELISA (blue) References 1. Crump JA, Luby SP, Mintz ED. The global burden of typhoid fever. Bull World Health Organ. 2004;82(5):346–53. 2. Hoffman SL, Edman DC, Punjabi NH, Lesmana M, Cholid A, Sundah S, et al. Bone Marrow Aspirate Culture Superior to Streptokinase Clot Culture and 8 ml 1:10 Blood-to-Broth Ratio Blood Culture for Diagnosis of Typhoid Fever. Am J Trop Med Hyg. 1986 Jul 1;35(4):836–9. 3. Taylor DN, Harris JR, Barrett TJ, Hargrett NT, Prentzel I, Valdivieso C, et al. Detection of urinary Vi antigen as a diagnostic test for typhoid fever. J Clin Microbiol. 1983;18(4):872–6. 4. Fadeel MA, Crump JA, Mahoney FJ, Nakhla IA, Mansour AM, Reyad B, et al. Rapid diagnosis of typhoid fever by enzyme-linked immunosorbent assay detection of Salmonella serotype Typhi antigens in urine. Am J Trop Med Hyg. 2004;70(3):323–8. 5. Waddington CS, Darton TC, Jones C, Haworth K, Peters A, John T, et al. An outpatient, ambulant-design, controlled human infection model using escalating doses of Salmonella Typhi challenge delivered in sodium bicarbonate solution. Clin Infect Dis. 2014;58(9):1230–40. Optical density (450 nm wavelength) Diagnosed Participants (n) Vi antigen detected in urine (n) Day 0 Day of diagnosis 21 0/21 0/21 Table 1 – Results on ELISA on participants urine from day 0 (pre-challenge) and day of diagnosis (either blood culture positive or temperature ≥ 38 o C for 12 hours)
